Broad Anti-Viral Capacities of Lian-Hua-Qing-Wen Capsule and Jin-Hua-Qing-Gan Granule and Rational use Against COVID-19 Based on Literature Mining

The novel coronavirus disease 2019 (COVID-19) has become a matter of international concern as the disease is spreading exponentially. Statistics showed that infected patients in China who received combined treatment of Traditional Chinese Medicine and modern medicine exhibited lower fatality rate and relatively better clinical outcomes. Both Lian-Hua-Qing-Wen Capsule (LHQWC) and Jin-Hua-Qing-Gan Granule (JHQGG) have been recommended by China Food and Drug Administration for the treatment of COVID-19 and have played a vital role in the prevention of a variety of viral infections. Here, we desired to analyze the broad-spectrum anti-viral capacities of LHQWC and JHQGG, and to compare their pharmacological functions for rational clinical applications. Based on literature mining, we found that both LHQWC and JHQGG were endowed with multiple antiviral activities by both targeting viral life cycle and regulating host immune responses and inflammation. In addition, from literature analyzed, JHQGG is more potent in modulating viral life cycle, whereas LHQWC exhibits better efficacies in regulating host anti-viral responses. When translating into clinical applications, oral administration of LHQWC could be more beneficial for patients with insufficient immune functions or for patients with alleviated symptoms after treatment with JHQGG.


INTRODUCTION
Lian-Hua-Qing-Wen Capsule and Jin-Hua-Qing-Gan Granule are Both Recommended as Effective "Chinese Solution" Against COVID-19 The novel coronavirus disease 2019  pandemics has reached almost every country in the world. Compared with the outbreak of Severe Acute Respiratory Syndrome (SARS) in 2003 and the pandemic of Middle East Respiratory Syndrome (MERS) in 2012, COVID-19 caused by the novel coronavirus SARS-CoV-2 infection has relatively low fatality rate, whereas much more rapid and higher human-to-human transmissibility (Meo et al., 2020). Typically, the existence of a large number of asymptomatic carriers of SARS-CoV-2 additionally exerts potential burden to the control and prevention of COVID-19.
SARS-CoV-2 can be easily transmitted through respiratory droplets or by aerosol, and infected people have a wide range of reported symptoms, from mild symptoms to severe illness. The most common manifestations of COVID-19 are fever or chill, dry cough and fatigue, which could be accompanied with a temporary loss of smell or taste, muscle or body aches. In critical cases, acute myocardial injury, liver or kidney dysfunction and blood-clotting complications may occur Huang et al. (2020), Khider et al. (2020), consequently leading to septic shock and acute respiratory distress syndrome (ARDS) or death. The "Clinical Treatment for COVID-19" issued by the World Health Organization recommends that symptomatic treatments that relieve fever and pain, together with adequate nutritional supports are basically required for mild cases of COVID-19. For severe SARS-CoV-2 infections, oxygen therapy and fluid supply need to be reinforced. In spite of supportive measures above, potential anti-viral drugs which were used for diseases due to viral infections other than SARS-CoV-2 have been repurposed for COVID-19, such as remdesivir, ribavirin and hydroxychloroquine are however not addressed because of reported side-effects or lack of supporting evidence from large-scale randomized controlled trials (Izcovich et al., 2020;Trivedi et al., 2020;Qaseem et al., 2021). Likewise, vaccine development involves a difficult, complex and costly process, and the success of which is at a high risk of failure protecting against mutant viral variants (Biswas and Majumder, 2020;Penarrubia et al., 2020). Despite the development of vaccines, scientists are still tirelessly designing new drugs and repurposing existing drugs against SARS-CoV-2. Though tremendous strides have been made in the fight against coronaviruses, a lack of safe and effective anti-SARS-CoV-2 drugs is still a key factor restricting the prevention and control of COVID-19 pandemics.
The practice of Traditional Chinese Medicine (TCM) has accumulated a wealth of clinical experience in the treatment of infectious diseases since Qin-Han (about 221 BC to 220 AD) and developed into a theory in Ming-Qing period (about 1,368-1777 AD). Infectious diseases in TCM have been described as "infections caused by toxic qi", "warm pathogen first invades lung via nose and mouth", and "disease spreads due to close contact". These descriptions fit well with the epidemiological characteristics of modern acute infectious diseases. According to TCM theory, COVID-19 is the result of invasion by dampnesstoxin pathogens, therefore COVID-19 is pathogenically characterized by dampness-toxin and host healthy-qi deficiency. Most patients first present mild sign of dampness, like fatigue, poor appetite and greasy thick tongue coating (Zheng, 2020). As disease progresses, dampness-toxin invades interiority and diffuses into triple energizer, leading to vital qi impairment and accumulation of toxin-qi in viscera. Excessive accumulation of dampness-toxin may easily lead to vital qi exhaustion and consequently loss of life. Hence, TCM formulae functioning to remove dampness-toxin are effective in preventing COVID-19 progress. Being the first country that was attacked by COVID-19, approximately 91.5% confirmed patients in China were treated with TCM formulae and the total effective rate has reached to 90%. In Wuhan Jiang-Xia Square Cabin Hospital, none of the 564 COVID-19 patients who received combined treatment of TCM and modern medicine developed into severe conditions, and TCM addition significantly reduced the course of hospitalization .
Both LHQWC and JHQGG belong to "Three Drugs, Three Prescriptions", official prescriptions of TCM used in the fight against COVID-19 in China. LHQWC, composed of Forsythia suspensa (Thunb.) Vahl, Lonicera japonica Thunb., honey-fried Ephedra sinica Stapf, fried Prunus sibirica L., Gypsum Fibrosum, Isatis tinctoria L., Dryopteris crassirhizoma Nakai, Houttuynia cordata Thunb., Pogostemon cablin (Blanco) Benth., Rheum palmatum L., Rhodiola crenulata (Hook.f. and Thomson) H. Ohba, Mentha canadensis L. and Glycyrrhiza glabra L., is innovative Chinese Patent Medicine (CPM) approved during the SARS epidemics in 2003. JHQGG, the other CPM constituting Forsythia suspensa (Thunb.) Vahl, Lonicera japonica Thunb., Ephedra sinica Stapf, Prunus sibirica L., l-Menthol, Glycyrrhiza glabra L., Scutellaria baicalensis Georgi, Fritillaria thunbergii Miq., Anemarrhena asphodeloides Bunge, Arctium lappa L. and Artemisia annua L., has been approved to treat H1N1 influenza virus infection since 2009. Both LHQWC and JHQGG are developed based on Ma-Xing-Shi-Gan Decoction and Yin-Qiao Powder, classic TCM decoctions used for respiratory infections recorded in Treatize on Exogenous Febrile Disease (about 210 AD) and Systematic Differentiation of Warm Diseases (1798 AD), respectively. In clinical practices resolving respiratory infections, LHQWC is mainly used to clear away plague, remove toxins, ventilate lungs and discharge heat, whereas JHQGG is applied to dispel wind, clear heat and resolve toxin. In the combat against COVID-19, National Health Commission of China approved both LHQWC and JHQGG as clinical therapies in China, and observational studies showed that both can effectively relieve fever, fatigue, cough and phlegm in the early stage of COVID-19, contributing to reductions in risks of rapid clinical deterioration. Supportively, in vitro studies have revealed that both formulae have anti-inflammatory effects, providing fundamental evidence for clinical application of both formulae in the fight against COVID-19 Duan, 2020;Runfeng et al., 2020;Zhang et al., 2020).
Holism Theory of TCM and Anti-viral Actions of Lian-Hua-Qing-Wen Capsule and Jin-Hua-Qing-Gan Granule, a Reflection of Host-Directed Therapy in Modern Medicine dovetails with the principle of host-directed therapy (HDT). HDT is a novel concept in the treatment for infectious diseases and was first used in tuberculosis in 2015 (Zumla et al., 2015). After then, HDT was gradually fulfilled as anti-viral strategies. Compared to conventional anti-viral therapies, which focus on inhibiting virus activity, HDT aims to maintain homeostasis of host by stimulating anti-viral responses and suppressing immune injuries. It has been shown that compared to single antipathogen treatment, HDT is able to reduce the risks of drug resistance induced by bacteria and viruses, endowing HDT a therapeutic potential of being broad-spectrum anti-viral tactics (Kaufmann et al., 2018). Clinical investigations proposed that viral infection-triggered cytokine storm was a vital factor mediating the rapid progress of COVID-19 (Wang T. et al., 2020). High levels of IL (Interleukin) -6 and IL-10, while low levels of CD4 + T and CD8 + T cells can be observed in COVID-19 patients Wan et al., 2020). Moreover, plasma IL-2, IL-7, IL-10, GCSF (granulocyte colony-stimulating factor), IP-10 (interferon gamma-induced protein-10), MCP-1 (monocyte chemoattractant protein-1), MIP-1α (macrophage inflammatory protein-1 alpha) and TNF-α (tumor necrosis factor-alpha) are consistently higher in intensive care unit (ICU) patients compared to mild cases , suggesting that virus-induced exaggerated immune responses and the resulting immune injuries are involved in the progression of COVID-19. Accordingly, HDT-oriented treatments that inhibit IL-6 signaling by down-regulating IL-6 receptors have been suggested as a potential solution for COVID-19 patients (Zumla et al., 2020). Consistent with HDT, in the combat against COVID-19, TCM addresses that sufficient healthy-qi within the body is key to prevent pathogen invasion, so-called "strengthening host resistance to eliminate pathogenic factors". Accordingly, inspiring vital qi is at the root of preventing infectious diseases in TCM. The functions of "healthy-qi" resemble "immunity" of host, and "pathogenic factors" stand for all substances that affect host homeostasis, such as viruses and bacteria. As emphasized in HDT that considering individuals as a whole rather than separating parts, "strengthening host resistance to eliminate pathogenic factors" in TCM addresses an overall reaction of host in response to invasive viruses, whereas the destiny of pathogen itself is not primarily important. Moreover, same as the HDT concept implicates, the ultimate goal of TCM treatment is to maintain host homeostasis via balancing interactions between host and pathogens, or by establishing equilibrium between stimulating anti-viral reactions and suppressing overactivated immune responses that subsequently cause tissue injuries.
Following the HDT principle and holism theory of TCM, this study primarily desired to gain more insight into the broad antiviral features of LHQWC and JHQGG, both of which have been applied to treat a variety of viral infections. However, considering that the main herbal composition of LHQWC and JHQGG largely overlap, it therefore appears confusing in the selection of appropriate formula for individual clinical cases. In this scenario, it is of prime importance to also distinguish the similarities and differences between the two formulae in terms of pharmacological anti-viral functions. To implement these goals, we manually grouped the individual active components from either LHQWC or JHQGG or both into two categories, namely constituents that interfere with viral life cycle and components that regulate host immune responses and inflammation. Through comprehensive literature review, data mining and pharmacological target enrichment analysis, we investigated the strength of LHQWC and JHQGG in the above-mentioned virus or host arm to compare their anti-viral functionalities. The holism-directed analysis of LHQWC and JHQGG will provide more insightful information and comprehensive understanding for rational use of these two CPMs in the combat against COVID-19, as well as the emerging or re-emerging pandemics of infectious diseases.

Literature Collection and Inclusion
In order to collect sufficient data on anti-viral effects of LHQWC and JHQGG, we employed Pubmed (https://pubmed.ncbi.nlm.nih. gov), Ovid (https://ovidsp.ovid.com/), CNKI (https://www.cnki. net), WANFANG (http://www.wanfangdata.com.cn/index.html) and WEIPU (http://www.cqvip.com/) database by searching either the full name of formulae, such as "Lianhua Qingwen Capsules", "Jinhua Qinggan Granules", or names of individual medicinal herbs, or active ingredients, together with "virus" as keywords. In addition, bioactive components that were proposed to be antivirals were included via network pharmacology-based prediction and analysis. A total of 1,110 articles were collected for next filtration. For the analysis of broad anti-viral activities, we then excluded studies reporting negative outcomes, clinical trials generally indicating viral infections without clarifying taxonomy of viruses, investigations using inactivated or attenuated viruses as vaccines, and articles with no access to full context due to age. A total of 812 articles were analyzed at this stage. For detailed comparisons of active anti-viral components and pharmacological functions of formulae, studies without indicating names of active components were further excluded. Notably, no information regarding Gypsum Fibrosum and fried Prunus sibirica L. in relevant to virus, and we did not find data by searching bioactive components directly isolated from JHQGG, hence we only took ingredients determined by predictive parsing of network pharmacology. Finally, 117 articles were included for comparison of pharmacological functions.

Classification of Viruses" Network
In order to describe broad-spectrum anti-viral activities of LHQWC and JHQGG, we grouped antiviral data collected as mentioned, and built a network in forms of "Formula-herb-virus-Baltimore classification of viruses". To further interpret the common and distinctive anti-viral activities of LHQWC and JHQGG in terms of holism theory of TCM, we classified the anti-viral actions reported for LHQWC and JHQGG into being either associated with viral life cycle or responsible to host immune responses and inflammation. To gain more insightful understanding, we further categorized active components that disrupt virus life cycle into three levels, including direct virucidal activity, inhibition of viral entry, and suppression of viral replication and egress. Generally, inhibitors of virus entry act through deforming viral particles or blocking the attachment or     binding of virions to host cells. The control of virus replication is mainly mediated by inhibiting replicator machineries encoded by viral systems, and prevention of virus egress is a process involves an interference with assembly and release of progeny viruses, which may initiate a secondary round infection. For the actions of regulating host immune responses and inflammation, it represents any virucidal effects due to an indirect response by modulating host immune system, such as increasing interferons (IFNs) expression, or decreasing self-targeted inflammatory injuries, or promoting repair process post virus infection without involving viral molecule-associated biological events. Based on literature mining and analysis, we next counted the frequencies of active components of LHQWC and JHQGG that have been sorted into each of the two categories, and accordingly a radar chart was drawn to visualize and compare the power of LHQWC and JHQGG against viral infection in terms of modulating viral life cycle and regulating host immune responses and inflammation.

RESULTS
The broad-Spectrum Anti-Viral Activities of Lian-Hua-Qing-Wen Capsule and Jin-Hua-Qing-Gan Granule Multi-ingredients, multi-targets and multi-pathways are primary features of TCM formulae, suggesting that active ingredients of one medicinal herb may exert anti-viral functions via diverse pharmacological mechanisms. As shown in Figure 1,

Similarities and Differences of Lian-Hua-Qing-Wen Capsule and Jin-Hua-Qing-Gan Granule as Antivirals
Both LHQWC and JHQGG possess broad-spectrum anti-viral potentials through interfering with viral life cycle and modulating host immune responses, which are associated with a diversity of proposed pharmacological actions as detailed in Tables 1, 2, 3; Figure 2. When comparing LHQWC and JHQGG, no difference was found in the types of their targeted viruses (Table 1; Figure 1). In terms of active components that disrupt viral life cycle (Table 1; Figure 2), only few literatures reported a direct virucidal activity from components of LHQWC and JHQGG (Table 1-1.1; Figure 2), about 24% studies showed suppression of viral entry (Table 1-1.2; Figure  2), while 70% studies focused on inhibitory effects toward viral replication and release (Table 1-1.3; Figure 2) Among all data analyzed, constituents from Scutellaria baicalensis Georgi (Huang Qin) of JHQGG have been mostly reported to interfere with viral life cycle in all three phases analyzed. Besides, components from Isatis tinctoria L (Ban Lan Gen) and Rheum palmatum L (Da Huang) of LHQWC are shown highly effective in blocking viral entry, replication and release. JHQGG weights slightly higher than LHQWC in terms of viral replication and release, whereas little difference was obtained in the early phase of viral life cycle (Table 1; Figure 2). Regarding "host immune responses and inflammation", it is interesting that constituents from Scutellaria baicalensis Georgi (Huang Qin) of JHQGG again exhibited the greatest potential, followed by components from Isatis tinctoria L (Ban Lan Gen) and Rheum palmatum L (Da Huang) in LHQWC. When comparing LHQWC and JHQGG, LHQWC weights slightly higher than JHQGG (Table 2; Figure 2). In addition, several studies have proposed other anti-viral mechanisms that could not be grouped into the above two categories, such as maintaining host redox homeostasis, or acting on microbiota, or gut-lung axis, or       Figure 2). Detailed information regarding the TCM features, pharmacological functions of individual herbs and components was outlined in Table 4.
In terms of COVID-19, the ACE-2 has been identified as the most important receptor for SARS-CoV-2 viral entry, which constitutes the initial step of infection (Walls et al., 2020). Through informatic analysis, the Rheum palmatum L (Da Huang) in LHQWC was found to be able to suppress viral infection by directly blocking interactions between the spike protein and ACE2. In addition, in the SARS-CoV, MERS-CoV and other coronaviruses, the 3CL (3C-like) protease is one of the crucial enzymes that mediates viral replication and has been recognized as a potential therapeutic target (Pillaiyar et al., 2016;Galasiti Kankanamalage et al., 2018). These predictive evaluations showed that Scutellaria baicalensis Georgi (Huang Qin), Anemarrhena asphodeloides Bunge (Zhi Mu) and Arctium lappa L (Niu Bang Zi) in JHQGG, as well as Rheum palmatum L (Da Huang) and Houttuynia cordata Thunb (Yu Xing Cao) in LHQWC can inhibit viral transcription and replication, especially that the Rheum palmatum L (Da Huang) in LHQWC was shown as a potential inhibitor of 3CL protease, suggesting underlying mechanisms of both LHQWC and JHQGG in the treatment of COVID-19.
Since LHQWC and JHQGG are both commonly used for the treatment of influenza in China, we additionally analyzed their possible roles in the inhibition of influenza viral invasion. Hemagglutinin (HA) on the surface of influenza virus is a tri-polymer, which promotes virus binding and entering into host cells. In contrast to HA, the neuraminidase (NA) of influenza viruses involves detachment and release of mature viruses from host cells (Gamblin and Skehel, 2010;Gaymard et al., 2016

DISCUSSION
In clinical practices of TCM, medicinal herbs are generally applied in the form of decoctions, which contain mixtures of a variety of herbs with different pharmacological functions. Instead of directly inactivating pathogens, therapeutic effects of TCM decoctions are achieved mainly through balancing host anti-viral responses and pathogenic factors. During COVID-19 epidemics, synergistic therapy of LHQWC with clinically approved reproposing antivirals, such as oseltamivir, umifenovir, ribavirin, lopinavir, peramivir, penciclovir or ganciclovir, has shown its advantages in improving associated symptoms and reducing the course of hospitalization and disease progression in several reported trials (Liu M. et al., 2020;Yu, 2020a;Yu, 2020b;Lv and Wang, 2020;Xiao et al., 2020;Liu et al., 2021). Similarly, combined anti-viral treatment with JHQGG in mild or moderate COVID-19 was beneficial in relieving clinical symptoms and reducing risks of severe COVID-19 (Liu Z. et al., 2020;Duan, 2020; Duan,        Ko et al. (2006) 2020). These studies provide clinical evidence that combined treatment with either LHQWC or JHQGG is superior to conventional monotherapy of antivirals. The primary conclusion of our study that both LHQWC and JHQGG are efficient for a large range of viral diseases has supported that TCM formulae can be potentially an alternative therapy for emerging viral diseases, especially when specific drugs and vaccines have not been fully developed and applied. However, when it comes to appropriate or precisive clinical applications of LHQWC and JHQGG, differences of their associated pharmacological actions turn out to be an essential point to be addressed. When comparing the anti-viral targets of LHQWC and JHQGG, both CPMs have been documented effective in interfering with viral components, with Isatis tinctoria L (Ban Lan Gen) and Rheum palmatum L (Da Huang) in LHQWC being the predominate viral inhibitors, followed by Lonicera japonica Thunb (Jin Yin Hua) and Houttuynia cordata Thunb (Yu Xing Cao). While in JHQGG, the Scutellaria baicalensis Georgi (Huang Qin) and subsequently Lonicera japonica Thunb (Jin Yin Hua) are the most important virucidal herbs. Typically, Scutellaria baicalensis Georgi (Huang Qin) of JHQGG have been highly nominated among all analyzed herbs contributing to suppression of the whole viral life cycle. Intriguingly, a direct virucidal activity was observed mostly in components from Scutellaria baicalensis Georgi (Huang Qin) and Anemarrhena asphodeloides Bunge (Zhi Mu) of JHQGG, though shared herbs, Lonicera japonica Thunb (Jin Yin Hua) and Glycyrrhiza glabra L (Gan Cao) were also involved. This set of data indicate that from the angle of viral life cycle, JHQGG may overweight LHQWC due to Scutellaria baicalensis Georgi (Huang Qin), and will be appropriate for patients with high fever, sore throat and cough. On the other hand, owning to existence of Rhodiola crenulata (Hook.f. and Thomson) H. Ohba (Hong Jing Tian), LHQWC may have more essential roles in the balancing of host immunity, suggesting that LHQWC could be more suitable for patients with non-efficient anti-viral immune responses.
There are some possible limitations in this study. Firstly, based on five databases, we finally included relatively more articles associated with LHQWC compared with those of JHQGG; therefore, bias could be unintendedly introduced to conclusions supporting superiority of LHQWC. Secondly, a certain number of included studies focus on Scutellaria baicalensis Georgi (Huang Qin), Isatis tinctoria L (Ban Lan Gen) and Rheum palmatum L (Da Huang); therefore, this may lead to biases that only these herbs are important as antivirals. Thirdly, the quality of articles included in this study is variable, and the judgment for potential pharmacological actions may to some degree rely on the knowledge of authors.
COVID-19 initiates with mild or moderate symptoms in most cases, and the strategy to reduce risks in evolving into severe or critical COVID-19 is highly desired. Through literature mining, we provide general evidence that both LHQWC and JHQGG are effective for mild to moderate COVID-19 patients and potentially being able to prevent the progress of COVID-19 into severe or critical conditions. As discussed above, TCM therapy fits well with the principle of HDT, and anti-viral TCM formulae generally show a broad spectrum of anti-viral properties through balancing between viral activities and host immune reactions. This has gained TCM a key advantage over target-specific anti-viral medications. Since LHQWC and JHQGG are both CPMs with clear safety information, it is imperative that application of LHQWC and JHQGG can be contextualized to worldwide combat against the emerging or re-emerging of human pandemics.

DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included in the article, further inquiries can be directed to the corresponding authors.

AUTHOR CONTRIBUTIONS
NL, RX and JL initiated and supervised this study. NL, RX, MS, BP, and AL performed data analysis and wrote this manuscript. PS assisted in organizing and analyzing data, and ZL contributed to editing.